Field of Science

Plasmid checking

Today I made it to the bench, to do plasmid preps to check that some gift plasmids had the expected structure. I needed to check this before freezing stocks of them in our lab strain collection. The plasmids contain fusions of E. coli CRP-S promoters to the E. coli lacZYA genes. I'll be using them as reporters to test my attempts to induce expression of the E. coli sxy gene. The genes are hofM (the E. coli homolog of H. influenzae's comA) and ppdA (the E. coli homolog of H. influenzae's pilB)

So I did minipreps using our nice Sigma kit, and digested the plasmids with a pair of enzymes that would give one of two patterns. I didn't know which of three restriction sites the inserts were in, so I could only predict two possible patterns from my digests (either one 11kb fragment, one 1.2kb fragment and tiny fragments of either ~300 or ~600 (hofM or ppdA); or one 11kb fragment and fragments of ~1.5kb and 1.8kb (hofM or ppdA).

But for the first time in my research career, I absentmindedly plugged the gel box electrodes in backwards - black cable into red connection and red into black. When I went back to check 25 minutes later, the tracking dye was running backwards out the top end of the gel. Rather than starting over, I just reversed the electrodes and let it run back the way it was supposed to go for a couple of hours, hoping that some information would be usable. Much to my surprise, the gel turned out great (the migration process must be perfectly reversible even though the DNA travelled twice through the wells). If the inserts had given the two tiny fragments they would have been lost during the backwards excursion, but luckily the cloning had generated the 1.5 and 1.8kb fragments instead.

1 comment:

  1. Ah, retro-phoresis. I made the same mistake in the last few months of my Ph.D work, which of course is the perfect time for such mistakes ;-) .


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